Protein purification

ABSTRACT

A method for purifying a polypeptide by ion exchange chromatography is described in which a gradient wash is used to resolve a polypeptide of interest from one or more contaminants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/659,825, filed Sep. 10, 2003 (now U.S. Pat. No. 8,044,017), which isa non-provisional application filed under 37 CFR 1.53(b)(1), claimingpriority under 35 USC §119(c) to U.S. Provisional Application No.60/410,334, filed Sep. 11, 2002, the entire contents of which areincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to protein purification. In particular,the invention relates to a method for purifying a polypeptide (e.g. anantibody) from a composition comprising the polypeptide and at least onecontaminant using the method of ion exchange chromatography.

2. Description of the Related Art

The large-scale, economic purification of proteins is an increasinglyimportant problem for the biotechnology industry. Generally, proteinsare produced by cell culture, using either eukaryotic or prokaryoticcell lines engineered to produce the protein of interest by insertion ofa recombinant plasmid containing the gene for that protein. Since thecells typically used are living organisms, they must be fed with acomplex growth medium, containing sugars, amino acids, and growthfactors, usually supplied from preparations of animal serum. Separationof the desired protein from the mixture of compounds fed to the cellsand from the by-products of the cells themselves to a purity sufficientfor use as a human therapeutic poses a formidable challenge.

Procedures for purification of proteins from cell debris initiallydepend on the site of expression of the protein. Some proteins can becaused to be secreted directly from the cell into the surrounding growthmedia; others are made intracellularly. For the latter proteins, thefirst step of a purification process involves lysis of the cell, whichcan be done by a variety of methods, including mechanical shear, osmoticshock, or enzymatic treatments. Such disruption releases the entirecontents of the cell into the homogenate, and in addition producessubcellular fragments that are difficult to remove due to their smallsize. These are generally removed by differential centrifugation or byfiltration. The same problem arises, although on a smaller scale, withdirectly secreted proteins due to the natural death of cells and releaseof intracellular host cell proteins in the course of the proteinproduction run.

Once a clarified solution containing the protein of interest has beenobtained, its separation from the other proteins produced by the cell isusually attempted using a combination of different chromatographytechniques. These techniques separate mixtures of proteins on the basisof their charge, degree of hydrophobicity, or size. Several differentchromatography resins are available for each of these techniques,allowing accurate tailoring of the purification scheme to the particularprotein involved. The essence of each of these separation methods isthat proteins can be caused either to move at different rates down along column, achieving a physical separation that increases as they passfurther down the column, or to adhere selectively to the separationmedium, being then differentially eluted by different solvents. In somecases, the desired protein is separated from impurities when theimpurities specifically adhere to the column, and the protein ofinterest does not, that is, the protein of interest is present in the“flow-through”.

Ion exchange chromatography is a chromatographic technique that iscommonly used for the purification of proteins. In ion exchangechromatography, charged patches on the surface of the solute areattracted by opposite charges attached to a chromatography matrix,provided the ionic strength of the surrounding buffer is low. Elution isgenerally achieved by increasing the ionic strength (i.e. conductivity)of the buffer to compete with the solute for the charged sites of theion exchange matrix. Changing the pH and thereby altering the charge ofthe solute is another way to achieve elution of the solute. The changein conductivity or pH may be gradual (gradient elution) or stepwise(step elution). In the past, these changes have been progressive; i.e.,the pH or conductivity is increased or decreased in a single direction

U.S. Pat. Nos. 6,339,142 and 6,417,355 (Basey et al.) describe ionexchange chromatography for purifying polypeptides.

U.S. Pat. Nos. 6,127,526 and 6,333,398 (Blank, G.) describe purifyingproteins, such as anti-HER2 antibodies, by Protein A chromatography.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method for purifying apolypeptide from a composition comprising the polypeptide andcontaminants. The composition is loaded onto an ion exchange resin withan equilibrium buffer having a first salt concentration. The ionexchange resin is washed with a wash buffer until a predeterminedprotein concentration is measured in the flowthrough. During the washthe salt concentration of the wash buffer increases from an initial,second salt concentration that is greater than the salt concentration ofthe equilibration buffer, to a final, third salt concentration. A fixedvolume of wash buffer at the final, third salt concentration is thenpassed over the resin. Finally, the polypeptide is eluted from the ionexchange resin exchange resin with elution buffer that has a saltconcentration that is greater than the final salt concentration of thewash buffer.

In one embodiment the ion exchange resin is an anion exchange resin. Inanother embodiment the ion exchange resin is a cation exchange resin.Preferably, the cation exchange resin comprises sulphopropyl immobilizedon agarose.

In another embodiment the elution buffer has a higher conductivity thanthe equilibration buffer. In a particular embodiment the elution buffercomprises about 145 mM Na/HOAc and the equilibration buffer comprisesabout 70 mM Na/HOAc. In another embodiment the elution buffer comprisesabout 100 mM NaCl and the equilibration buffer comprises about 45 mMNaCl.

The wash buffer preferably comprises a mixture of equilibration bufferand elution buffer. Thus, in one embodiment the increase in the saltconcentration of the wash buffer during step (b) is achieved byincreasing the proportion of elution buffer in the wash buffer. Theproportion of elution buffer in the wash buffer may be increased at aconstant rate. In one embodiment the increase in the proportion ofelution buffer causes the salt concentration of the wash buffer toincrease at a constant rate of from about 1 mM to about 3 mM per columnvolume of wash buffer.

In another embodiment the percentage of elution buffer in the washbuffer increases at two or more different rates during the course ofwashing in step (b). For example, the percentage of elution buffer inthe wash buffer increases at a first rate for a first segment of thewashing, at a second rate for a second segment of the washing and at athird rate for a third segment of the washing.

In one embodiment the polypeptide that is purified is an antibody. Inthis case, the contaminant may be a deamidated variant of the antibody.In a particular embodiment, the antibody binds HER2. In one embodimentthe amount of antibody in the composition loaded onto the ion exchangeresin is from about 15 mg to about 45 mg per mL of cation exchangeresin.

In one embodiment the predetermined protein concentration corresponds toan OD of 0.6 measured at 280 nm. In another embodiment, from about 0.4to about 1 column volumes of wash buffer are passed over the ionexchange resin in step (c). In a further embodiment the pH of theequilibration buffer, wash buffer and elution buffer is approximatelythe same, preferably approximately 5.5.

In another embodiment the method of purifying a polypeptide furthercomprises subjecting the composition comprising the polypeptide to oneor more additional purification steps, so as to obtain a homogeneouspreparation of the polypeptide. In a further embodiment, apharmaceutical composition is prepared by combining the homogeneouspreparation of the polypeptide with a pharmaceutically acceptablecarrier. In another embodiment the purified polypeptide is conjugatedwith a heterologous molecule, for example polyethylene glycol, a labelor a cytotoxic agent.

In another aspect, the invention provides a polypeptide which has beenpurified according to the method provided herein.

In a further aspect, the invention provides a method for purifying anantibody from a composition comprising the polypeptide and acontaminant. The antibody is bound to a cation exchange material with anequilibration buffer at a first conductivity. The cation exchangematerial is washed with a wash buffer, wherein the conductivity of thewash buffer increases from a second conductivity that is higher than thefirst conductivity to a third conductivity during the washing. A fixedvolume of wash buffer at the third conductivity is then passed over thecation exchange material and the antibody is eluted from the cationexchange material with an elution buffer at a fourth conductivity thatis higher than the third conductivity. The cation exchange resinpreferably comprises sulphopropyl immobilized on agarose. In oneembodiment the fixed volume of wash buffer passed over the cationexchange material is between about 0.4 column volumes and about 1.0column volumes. The method may also include the step of washing the ionexchange material with a regeneration buffer following elution of theantibody.

In one embodiment the conductivity of the wash buffer increases at aconstant rate from the second conductivity to the third conductivity,while in another embodiment the conductivity of the wash bufferincreases at two or more different rates from the second conductivity tothe third conductivity. In a particular embodiment the conductivity ofthe wash buffer increases at a first rate for a first segment of thewashing, at a second rate for a second segment of the washing and at athird rate for a third segment of the washing.

Preferably the wash buffer comprises a mixture of equilibration bufferand elution buffer. In this case, the conductivity of the wash buffermay be increased by increasing the proportion of elution buffer in thewash buffer. In one embodiment the proportion of elution buffer in thewash buffer increases at a constant rate of about 6% during the firstsegment, at a constant rate of about 3.5% during the second segment andat a constant rate of about 2% during the third segment. In anotherembodiment the proportion of elution buffer in the wash buffer increasesfrom about 26% to about 54% during the first segment, from about 54% toabout 61% during the second segment and from about 61% to about 74%during the second segment.

In a further embodiment the cation exchange material is washed withabout 5 column volumes of wash buffer in the first segment, about 2column volumes of wash buffer in the second segment and about 6 columnvolumes of wash buffer in the third segment.

The conductivity of the wash buffer may be increased by increasing thepercentage of elution buffer in the wash buffer. In another embodimentthe conductivity of the wash buffer is increased by increasing the saltconcentration therein.

In a further aspect, the invention provides a method for purifying anantibody from a composition comprising the antibody and a contaminant.Preferably the composition is loaded onto a cation exchange material,the cation exchange material is washed with a wash buffer with aconductivity that increases at a first rate from a first conductivity toa second conductivity, at a second rate from the second conductivity toa third conductivity and at a third rate from the third conductivity toa fourth conductivity, and the antibody is eluted from the ion exchangematerial. The amount of antibody in the composition loaded onto thecation exchange material is preferably from about 15 mg to about 45 mgof the antibody per ml of cation exchange material.

In a further aspect, the present invention provides a method forpurifying a polypeptide from a composition comprising the polypeptideand a contaminant comprising loading the composition onto an ionexchange material, washing the cation exchange material with wash bufferusing a multi-slope gradient until a predetermined protein concentrationis measured in the flowthrough, and eluting the polypeptide from the ionexchange material.

In one embodiment the multi-slope gradient comprises two or moresegments. Preferably, each segment of the multi-slope gradient has ashallower slope.

In another embodiment the method additionally comprises the step ofwashing the column with from 0.4 to 1 column volumes of wash bufferafter the multi-slope gradient wash and prior to eluting thepolypeptide. Preferably the wash buffer used in this additional step hasthe composition of the wash buffer at the end of the multi-slopegradient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show the amino acid sequences of humMAb4D5-8 light chain(SEQ ID NO:1) and humMAb4D5-8 heavy chain (SEQ ID NO:2), respectively.

FIG. 2 is a graph illustrating a chromatography process with a lineargradient wash step. By testing gradients of various slopes (1-4 mMNaCl/CV), we found that although the yield and purity were moreconsistent at different loads than when a step wash procedure was used,there was still more variability than desired.

FIG. 3 is a graph illustrating a chromatography process with amulti-slope gradient wash. By using a linear gradient with threesegments (having progressively shallower slopes), consistent yield andpurity can be achieved across the entire load range. In addition, asshown below, the trade off between yield and purity can be fine tuned byadjusting the end-wash delay volume after OD of 0.6 is achieved.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Definitions

The “composition” to be purified herein comprises the polypeptide ofinterest and one or more contaminants. The composition may be “partiallypurified” (i.e. having been subjected to one or more purification steps,such as Protein A Chromatography) or may be obtained directly from ahost cell or organism producing the polypeptide (e.g. the compositionmay comprise harvested cell culture fluid).

As used herein, “polypeptide” refers generally to peptides and proteinshaving more than about ten amino acids. Preferably, the polypeptide is amammalian protein, examples of which include: renin; a growth hormone,including human growth hormone and bovine growth hormone; growth hormonereleasing factor; parathyroid hormone; thyroid stimulating hormone;lipoproteins; alpha-1-antitrypsin; insulin A-chain; insulin B-chain;proinsulin; follicle stimulating hormone; calcitonin; luteinizinghormone; glucagon; clotting factors such as factor VIIIC, factor IX,tissue factor, and von Willebrands factor; anti-clotting factors such asProtein C; atrial natriuretic factor; lung surfactant; a plasminogenactivator, such as urokinase or human urine or tissue-type plasminogenactivator (t-PA); bombesin; thrombin; hemopoietic growth factor; tumornecrosis factor-alpha and -beta; enkephalinase; RANTES (regulated onactivation normally T-cell expressed and secreted); human macrophageinflammatory protein (MIP-1-alpha); a serum albumin such as human serumalbumin; Muellerian-inhibiting substance; relaxin A-chain; relaxinB-chain; prorelaxin; mouse gonadotropin-associated peptide; a microbialprotein, such as beta-lactamase; DNase; IgE; a cytotoxic T-lymphocyteassociated antigen (CTLA), such as CTLA-4; inhibin; activin; vascularendothelial growth factor (VEGF); receptors for hormones or growthfactors; Protein A or D; rheumatoid factors; a neurotrophic factor suchas bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or-6 (NT-3, NT-4, NT-5, or NT-6), or a nerve growth factor such as NGF-β;platelet-derived growth factor (PDGF); fibroblast growth factor such asaFGF and bFGF; epidermal growth factor (EGF); transforming growth factor(TGF) such as TGF-alpha and TGF-beta, including TGF-β1, TGF-β2, TGF-β3,TGF-β4, or TGF-β5; insulin-like growth factor-I and -II (IGF-I andIGF-II); des(1-3)-IGF-I (brain IGF-I), insulin-like growth factorbinding proteins (IGFBPs); CD proteins such as CD3, CD4, CD8, CD19 andCD20; erythropoietin; osteoinductive factors; immunotoxins; a bonemorphogenetic protein (BMP); an interferon such as interferon-alpha,-beta, and -gamma; colony stimulating factors (CSFs), e.g., M-CSF,GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-10; superoxidedismutase; T-cell receptors; surface membrane proteins; decayaccelerating factor; viral antigen such as, for example, a portion ofthe AIDS envelope; transport proteins; homing receptors; addressins;regulatory proteins; integrins such as CD11a, CD11b, CD11c, CD18, anICAM, VLA-4 and VCAM; a tumor associated antigen such as HER2, HER3 orHER4 receptor; and fragments and/or variants of any of the above-listedpolypeptides. Most preferred is a full-length antibody that binds humanHER2.

A “contaminant” is a material that is different from the desiredpolypeptide product. The contaminant may be, without limitation, avariant, fragment, aggregate or derivative of the desired polypeptide(e.g. a deamidated variant or an amino-aspartate variant), anotherpolypeptide, nucleic acid, endotoxin, etc.

A “variant” or “amino acid sequence variant” of a starting polypeptideis a polypeptide that comprises an amino acid sequence different fromthat of the starting polypeptide. Generally, a variant will possess atleast 80% sequence identity, preferably at least 90% sequence identity,more preferably at least 95% sequence identity, and most preferably atleast 98% sequence identity with the native polypeptide. Percentagesequence identity is determined, for example, by the Fitch et al., Proc.Natl. Acad. Sci. USA 80:1382-1386 (1983), version of the algorithmdescribed by Needleman et al., J. Mol. Biol. 48:443-453 (1970), afteraligning the sequences to provide for maximum homology. Amino acidsequence variants of a polypeptide may be prepared by introducingappropriate nucleotide changes into DNA encoding the polypeptide, or bypeptide synthesis. Such variants include, for example, deletions from,and/or insertions into and/or substitutions of, residues within theamino acid sequence of the polypeptide of interest. Any combination ofdeletion, insertion, and substitution is made to arrive at the finalconstruct, provided that the final construct possesses the desiredcharacteristics. The amino acid changes also may alterpost-translational processing of the polypeptide, such as by changingthe number or position of glycosylation sites. Other post-translationalmodifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl, threonyl or tyrosylresidues, methylation of the α-amino groups of lysine, arginine andhistidine side chains (T. E. Creighton, Proteins: Structure andMolecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86(1983)). Methods for generating amino acid sequence variants ofpolypeptides are described in U.S. Pat. No. 5,534,615, expresslyincorporated herein by reference, for example.

An “acidic variant” is a variant of a polypeptide of interest which ismore acidic (e.g. as determined by cation exchange chromatography) thanthe polypeptide of interest. Acidic variants may be produced by theaction of recombinant host cells on the expressed polypeptide. Anexample of an acidic variant is a deamidated variant. Glutaminyl andaspariginyl residues are frequently post-translationally deamidated tothe corresponding glutamyl and aspartyl residues.

A “deamidated” variant of a polypeptide molecule is a polypeptidewherein one or more asparagine residue(s) of the original polypeptidehave been converted to aspartate, i.e. the neutral amide side chain hasbeen converted to a residue with an overall acidic character. Forexample, “deamidated human DNase” as used herein means human DNase thatis deamidated at the asparagine residue that occurs at position 74 inthe amino acid sequence of native mature human DNase (U.S. Pat. No.5,279,823; expressly incorporated herein by reference). DeamidatedhuMAb4D5 antibody from the Examples below has Asn30 in CDR1 of either orboth of the V_(L) regions thereof converted to aspartate.

In preferred embodiments of the invention, the polypeptide is arecombinant polypeptide. A “recombinant polypeptide” is one which hasbeen produced in a host cell which has been transformed or transfectedwith nucleic acid encoding the polypeptide, or produces the polypeptideas a result of homologous recombination. “Transformation” and“transfection” are used interchangeably to refer to the process ofintroducing nucleic acid into a cell. Following transformation ortransfection, the nucleic acid may integrate into the host cell genome,or may exist as an extrachromosomal element. The “host cell” includes acell in in vitro cell culture as well as a cell within a host animal.Methods for recombinant production of polypeptides are described in U.S.Pat. No. 5,534,615, expressly incorporated herein by reference, forexample.

The term “antibody” is used in the broadest sense and specificallycovers monoclonal antibodies (including full length monoclonalantibodies), polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired biological activity.

The antibody herein is directed against an “antigen” of interest.Preferably, the antigen is a biologically important polypeptide andadministration of the antibody to a mammal suffering from a disease ordisorder can result in a therapeutic benefit in that mammal. However,antibodies directed against non-polypeptide antigens (such astumor-associated glycolipid antigens; see U.S. Pat. No. 5,091,178) arealso contemplated. Where the antigen is a polypeptide, it may be atransmembrane molecule (e.g. receptor) or ligand such as a growthfactor. Exemplary antigens include those polypeptides discussed above.Preferred molecular targets for antibodies encompassed by the presentinvention include CD polypeptides such as CD3, CD4, CD8, CD19, CD20 andCD34; members of the HER receptor family such as the EGF receptor, HER2,HER3 or HER4 receptor; cell adhesion molecules such as LFA-1, Mac1,p150, 95, VLA-4, ICAM-1, VCAM and av/b3 integrin including either a or bsubunits thereof (e.g. anti-CD11a, anti-CD18 or anti-CD11b antibodies);growth factors such as VEGF; IgE; blood group antigens; flk2/flt3receptor; obesity (OB) receptor; mpl receptor; CTLA-4; polypeptide Cetc. Soluble antigens or fragments thereof, optionally conjugated toother molecules, can be used as immunogens for generating antibodies.For transmembrane molecules, such as receptors, fragments of these (e.g.the extracellular domain of a receptor) can be used as the immunogen.Alternatively, cells expressing the transmembrane molecule can be usedas the immunogen. Such cells can be derived from a natural source (e.g.cancer cell lines) or may be cells which have been transformed byrecombinant techniques to express the transmembrane molecule.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. The modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler et al., Nature256:495 (1975), or may be made by recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567). In a further embodiment, “monoclonalantibodies” can be isolated from antibody phage libraries generatedusing the techniques described in McCafferty et al., Nature, 348:552-554(1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J.Mol. Biol., 222:581-597 (1991) describe the isolation of murine andhuman antibodies, respectively, using phage libraries. Subsequentpublications describe the production of high affinity (nM range) humanantibodies by chain shuffling (Marks et al., Bio/Technology, 10:779-783(1992)), as well as combinatorial infection and in vivo recombination asa strategy for constructing very large phage libraries (Waterhouse etal., Nuc. Acids. Res., 21:2265-2266 (1993)). Thus, these techniques areviable alternatives to traditional monoclonal antibody hybridomatechniques for isolation of monoclonal antibodies. Alternatively, it isnow possible to produce transgenic animals (e.g., mice) that arecapable, upon immunization, of producing a full repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy-chain joining region (J_(H)) gene in chimeric andgerm-line mutant mice results in complete inhibition of endogenousantibody production. Transfer of the human germ-line immunoglobulin genearray in such germ-line mutant mice will result in the production ofhuman antibodies upon antigen challenge. See, e.g., Jakobovits et al.,Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature,362:255-258 (1993); Bruggermann et al., Year in Immuno., 7:33 (1993);and Duchosal et al. Nature 355:258 (1992).

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region comprises amino acid residues from a“complementarity determining region” or “CDR” (i.e. residues 24-34 (L1),50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35(H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain;Kabat et al., Sequences of Polypeptides of Immunological Interest, 5thEd. Public Health Service, National Institutes of Health, Bethesda, Md.(1991)) and/or those residues from a “hypervariable loop” (i.e. residues26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domainand 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). “Framework”or “FR” residues are those variable domain residues other than thehypervariable region residues as herein defined. The CDR and FR residuesof the rhuMAb HER2 antibody of the example below (humAb4D5-8) areidentified in Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, Fv framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues which are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin sequence. Thehumanized antibody optionally also will comprise at least a portion ofan immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework (FR) for the humanized antibody (Sims et al., J.Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901(1987)).

Another method uses a particular framework derived from the consensussequence of all human antibodies of a particular subgroup of light orheavy chains. The same framework may be used for several differenthumanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285(1992); Presta et al., J. Immunol., 151:2623 (1993)).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a preferred method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.

“Antibody fragments” comprise a portion of a full length antibody,generally the antigen binding or variable region thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂, and Fv fragments;diabodies; linear antibodies; single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments. Varioustechniques have been developed for the production of antibody fragments.Traditionally, these fragments were derived via proteolytic digestion ofintact antibodies (see, e.g., Morimoto et al., Journal of Biochemicaland Biophysical Methods 24:107-117 (1992) and Brennan et al., Science,229:81 (1985)). However, these fragments can now be produced directly byrecombinant host cells. For example, the antibody fragments can beisolated from the antibody phage libraries discussed above.Alternatively, Fab′-SH fragments can be directly recovered from E. coliand chemically coupled to form F(ab′)₂ fragments (Carter et al.,Bio/Technology 10:163-167 (1992)). In another embodiment, the F(ab′)₂ isformed using the leucine zipper GCN4 to promote assembly of the F(ab′)₂molecule. According to another approach, F(ab′)₂ fragments can beisolated directly from recombinant host cell culture. Other techniquesfor the production of antibody fragments will be apparent to the skilledpractitioner.

In other embodiments, the antibody of choice is a single chain Fvfragment (scFv). See WO 93/16185. “Single-chain Fv” or “sFv” antibodyfragments comprise the V_(H) and V_(L) domains of antibody, whereinthese domains are present in a single polypeptide chain. Generally, theFv polypeptide further comprises a polypeptide linker between the V_(H)and V_(L) domains which enables the sFv to form the desired structurefor antigen binding. For a review of sFv see Pluckthun in ThePharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Mooreeds. Springer-Verlag, New York, pp. 269-315 (1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy chain variabledomain (V_(H)) connected to a light chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993).

The expression “linear antibodies” when used throughout this applicationrefers to the antibodies described in Zapata et al. Polypeptide Eng.8(10):1057-1062 (1995). Briefly, these antibodies comprise a pair oftandem Fd segments (V_(H)-C_(H)1-V_(H)-C_(H)1) which form a pair ofantigen binding regions. Linear antibodies can be bispecific ormonospecific.

“Multispecific antibodies” have binding specificities for at least twodifferent epitopes, where the epitopes are usually from differentantigens. While such molecules normally will only bind two antigens(i.e. bispecific antibodies, BsAbs), antibodies with additionalspecificities such as trispecific antibodies are encompassed by thisexpression when used herein. Examples of BsAbs include those with onearm directed against a tumor cell antigen and the other arm directedagainst a cytotoxic trigger molecule such as anti-FcγRI/anti-CD15,anti-p185^(HER2)/FcγRIII (CD16), anti-CD3/anti-malignant B-cell (1D10),anti-CD3/anti-p185^(HER2), anti-CD3/anti-p97, anti-CD3/anti-renal cellcarcinoma, anti-CD3/anti-OVCAR-3, anti-CD3/L-D1 (anti-colon carcinoma),anti-CD3/anti-melanocyte stimulating hormone analog, anti-EGFreceptor/anti-CD3, anti-CD3/anti-CAMA1, anti-CD3/anti-CD19,anti-CD3/MoV18, anti-neural cell ahesion molecule (NCAM)/anti-CD3,anti-folate binding protein (FBP)/anti-CD3, anti-pan carcinomaassociated antigen (AMOC-31)/anti-CD3; BsAbs with one arm which bindsspecifically to a tumor antigen and one arm which binds to a toxin suchas anti-saporin/anti-Id-1, anti-CD22/anti-saporin,anti-CD7/anti-saporin, anti-CD38/anti-saporin, anti-CEA/anti-ricin Achain, anti-interferon-α(IFN-α)/anti-hybridoma idiotype,anti-CEA/anti-vinca alkaloid; BsAbs for converting enzyme activatedprodrugs such as anti-CD30/anti-alkaline phosphatase (which catalyzesconversion of mitomycin phosphate prodrug to mitomycin alcohol); BsAbswhich can be used as fibrinolytic agents such as anti-fibrin/anti-tissueplasminogen activator (tPA), anti-fibrin/anti-urokinase-type plasminogenactivator (uPA); BsAbs for targeting immune complexes to cell surfacereceptors such as anti-low density lipoprotein (LDL)/anti-Fc receptor(e.g. FcγRI, or FcγRIII); BsAbs for use in therapy of infectiousdiseases such as anti-CD3/anti-herpes simplex virus (HSV), anti-T-cellreceptor:CD3 complex/anti-influenza, anti-FcγR/anti-HIV; BsAbs for tumordetection in vitro or in vivo such as anti-CEA/anti-EOTUBE,anti-CEA/anti-DPTA, anti-p185^(HER2)/anti-hapten; BsAbs as vaccineadjuvants; and BsAbs as diagnostic tools such as anti-rabbitIgG/anti-ferritin, anti-horse radish peroxidase (HRP)/anti-hormone,anti-somatostatin/anti-substance P, anti-HRP/anti-FITC,anti-CEA/anti-β-galactosidase. Examples of trispecific antibodiesinclude anti-CD3/anti-CD4/anti-CD37, anti-CD3/anti-CD5/anti-CD37 andanti-CD3/anti-CD8/anti-CD37. Bispecific antibodies can be prepared asfull length antibodies or antibody fragments (e.g. F(ab′)₂ bispecificantibodies).

Methods for making bispecific antibodies are known in the art.Traditional production of full-length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature, 305:537-539 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, and in Traunecker et al., EMBOJ., 10:3655-3659 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion preferablyis with an immunoglobulin heavy chain constant domain, comprising atleast part of the hinge, CH2, and CH3 regions. It is preferred to havethe first heavy-chain constant region (CH1) containing the sitenecessary for light chain binding, present in at least one of thefusions. DNAs encoding the immunoglobulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable hostorganism. This provides for great flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yields. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains in oneexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios are of noparticular significance.

In a preferred embodiment of this approach, the bispecific antibodiesare composed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies see, for example, Suresh etal., Methods in Enzymology, 121:210 (1986).

According to another approach described in WO96/27011, the interfacebetween a pair of antibody molecules can be engineered to maximize thepercentage of heterodimers which are recovered from recombinant cellculture. The preferred interface comprises at least a part of the C_(H)3domain of an antibody constant domain. In this method, one or more smallamino acid side chains from the interface of the first antibody moleculeare replaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science, 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)₂ fragments. Thesefragments are reduced in the presence of the dithiol complexing agentsodium arsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)₂molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See Gruber et al., J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60(1991).

The phrase “ion exchange material” refers to a solid phase that isnegatively charged (i.e. a cation exchange resin) or positively charged(i.e. an anion exchange resin). The charge may be provided by attachingone or more charged ligands to the solid phase, e.g. by covalentlinking. Alternatively, or in addition, the charge may be an inherentproperty of the solid phase (e.g. as is the case for silica, which hasan overall negative charge).

By “solid phase” is meant a non-aqueous matrix to which one or morecharged ligands can adhere. The solid phase may be a purificationcolumn, a discontinuous phase of discrete particles, a membrane, orfilter etc. Examples of materials for forming the solid phase includepolysaccharides (such as agarose and cellulose) and other mechanicallystable matrices such as silica (e.g. controlled pore glass),poly(styrenedivinyl)benzene, polyacrylamide, ceramic particles andderivatives of any of the above.

A “cation exchange resin” refers to a solid phase that is negativelycharged and has free cations for exchange with cations in an aqueoussolution passed over or through the solid phase. A negatively chargedligand attached to the solid phase to form the cation exchange resinmay, e.g., be a carboxylate or sulfonate. Commercially available cationexchange resins include carboxy-methyl-cellulose, BAKERBOND ABX™,sulphopropyl (SP) immobilized on agarose (e.g. SP-SEPHAROSE FAST FLOW™,SP-SEPHAROSE FAST FLOW XL™ or SP-SEPHAROSE HIGH PERFORMANCE™, fromPharmacia) and sulphonyl immobilized on agarose (e.g. S-SEPHAROSE FASTFLOW™ from Pharmacia).

The term “anion exchange resin” is used herein to refer to a solid phasewhich is positively charged, e.g. having one or more positively chargedligands, such as quaternary amino groups, attached thereto. Commerciallyavailable anion exchange resins include DEAE cellulose, QAE SEPHADEX™and FAST Q SEPHAROSE™ (Pharmacia).

A “buffer” is a solution that resists changes in pH by the action of itsacid-base conjugate components. Various buffers which can be employeddepending, for example, on the desired pH of the buffer are described inBuffers. A Guide for the Preparation and Use of Buffers in BiologicalSystems, Gueffroy, D., Ed. Calbiochem Corporation (1975). In oneembodiment, the buffer has a pH in the range from about 5 to about 7(e.g. as in Example 1 below). Examples of buffers that will control thepH in this range include MES, MOPS, MOPSO, phosphate, acetate, citrate,succinate, and ammonium buffers, as well as combinations of these. Thebuffers used in the disclosed methods typically also comprise a salt,such as NaCL, KCl or NaHOAc.

“Equilibration buffer” is the buffer that is used to equilibrate the ionexchange resin. The equilibration buffer may also be used to load thecomposition comprising the polypeptide molecule of interest and one ormore contaminants onto the ion exchange resin. The equilibration bufferpreferably has a conductivity and/or pH such that the polypeptidemolecule of interest is bound to the ion exchange resin.

The term “wash buffer” is used herein to refer to the buffer that ispassed over the ion exchange resin following loading and prior toelution of the protein of interest. The wash buffer may serve to eluteone or more contaminants from the ion exchange resin. The conductivityand/or pH of the wash buffer is/are such that the contaminants areeluted from the ion exchange resin, but not significant amounts of thepolypeptide of interest. The “wash buffer” preferably comprises amixture of equilibration buffer and elution buffer, and can thus bedescribed by the percentage of elution buffer that it comprises in agiven volume.

“Elution buffer” is used to elute the polypeptide of interest from thesolid phase. The conductivity and/or pH of the elution buffer is/aresuch that the polypeptide of interest is eluted from the ion exchangeresin.

A “regeneration buffer” may be used to regenerate the ion exchange resinsuch that it can be re-used. The regeneration buffer has a conductivityand/or pH as required to remove substantially all contaminants and thepolypeptide of interest from the ion exchange resin.

The term “conductivity” refers to the ability of an aqueous solution toconduct an electric current between two electrodes. In solution, thecurrent flows by ion transport. Therefore, with an increasing amount ofions present in the aqueous solution, the solution will have a higherconductivity. The unit of measurement for conductivity is mmhos (mS/cm),and can be measured using a conductivity meter, such as those sold,e.g., by Orion. The conductivity of a solution may be altered bychanging the concentration of ions therein. For example, theconcentration of a buffering agent and/or the concentration of a salt(e.g. Na/HOAc, NaCl or KCl) in the solution may be altered in order toachieve the desired conductivity. Preferably, the salt concentration ofthe various buffers is modified to achieve the desired conductivity.

By “purifying” a polypeptide from a composition comprising thepolypeptide and one or more contaminants is meant increasing the degreeof purity of the polypeptide in the composition by removing (completelyor partially) at least one contaminant from the composition. A“purification step” may be part of an overall purification processresulting in a “homogeneous” composition. “Homogeneous” is used hereinto refer to a composition comprising at least about 70% by weight of thepolypeptide of interest, based on total weight of the composition,preferably at least about 80% by weight, more preferably at least about90% by weight, even more preferably at least about 95% by weight.

Unless indicated otherwise, the term “HER2” when used herein refers tohuman HER2 protein and “HER2” refers to human HER2 gene. The human HER2gene and HER2 protein are described in Semba et al., PNAS (USA)82:6497-6501 (1985) and Yamamoto et al. Nature 319:230-234 (1986)(Genebank accession number X03363), for example.

The term “huMAb4D5-8” when used herein refers to a humanized anti-HER2antibody comprising the light chain amino acid sequence of SEQ ID NO:1and the heavy chain amino acid sequence of SEQ ID NO:2 or amino acidsequence variants thereof which retain the ability to bind HER2 andinhibit growth of tumor cells which overexpress HER2 (see U.S. Pat. No.5,677,171; expressly incorporated herein by reference).

The “pI” or “isoelectric point” of a polypeptide refers to the pH atwhich the polypeptide's positive charge balances its negative charge. pIcan be calculated from the net charge of the amino acid residues of thepolypeptide or can be determined by isoelectric focussing.

By “binding” a molecule to an ion exchange material is meant exposingthe molecule to the ion exchange material under appropriate conditions(pH/conductivity) such that the molecule is reversibly immobilized in oron the ion exchange material by virtue of ionic interactions between themolecule and a charged group or charged groups of the ion exchangematerial.

By “washing” the ion exchange material is meant passing an appropriatebuffer through or over the ion exchange material.

To “elute” a molecule (e.g. polypeptide or contaminant) from an ionexchange material is meant to remove the molecule therefrom by alteringthe ionic strength of the buffer surrounding the ion exchange materialsuch that the buffer competes with the molecule for the charged sites onthe ion exchange material.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadywith the disorder as well as those in which the disorder is to beprevented.

A “disorder” is any condition that would benefit from treatment with thepolypeptide purified as described herein. This includes both chronic andacute disorders and diseases and those pathological conditions whichpredispose the mammal to the disorder in question.

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to thepolypeptide. The label may be itself be detectable (e.g., radioisotopelabels or fluorescent labels) or, in the case of an enzymatic label, maycatalyze chemical alteration of a substrate compound or compositionwhich is detectable.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g. I¹³¹,I¹²⁵, Y⁹⁰ and Re¹⁸⁶), chemotherapeutic agents, and toxins such asenzymatically active toxins of bacterial, fungal, plant or animalorigin, or fragments thereof.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includeadriamycin, doxorubicin, epirubicin, 5-fluorouracil, cytosinearabinoside (“Ara-C”), cyclophosphamide, thiotepa, busulfan, cytoxin,taxoids, e.g. paclitaxel (TAXOL™, Bristol-Myers Squibb Oncology,Princeton, N.J.), and doxetaxel, toxotere, methotrexate, cisplatin,melphalan, vinblastine, bleomycin, etoposide, ifosfamide, mitomycin C,mitoxantrone, vincristine, vinorelbine, carboplatin, teniposide,daunomycin, caminomycin, aminopterin, dactinomycin, mitomycins,esperamicins (see U.S. Pat. No. 4,675,187), melphalan and other relatednitrogen mustards. Also included in this definition are hormonal agentsthat act to regulate or inhibit hormone action on tumors, such astamoxifen and onapristone.

Modes For Carrying Out The Invention

The invention herein provides methods for purifying a polypeptide from acomposition (e.g. an aqueous solution) comprising the polypeptide andone or more contaminants. The composition is generally one resultingfrom the recombinant production of the polypeptide, but may be thatresulting from production of the polypeptide by peptide synthesis (orother synthetic means) or the polypeptide may be purified from a nativesource of the polypeptide. Preferably the polypeptide is an antibody,e.g. one which binds the HER2 antigen.

For recombinant production of the polypeptide, the nucleic acid encodingit is isolated and inserted into a replicable vector for further cloning(amplification of the DNA) or for expression. DNA encoding thepolypeptide is readily isolated and sequenced using conventionalprocedures (e.g., where the polypeptide is an antibody by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of the antibody). Many vectors areavailable. The vector components generally include, but are not limitedto, one or more of the following: a signal sequence, an origin ofreplication, one or more marker genes, an enhancer element, a promoter,and a transcription termination sequence (e.g. as described in U.S. Pat.No. 5,534,615, specifically incorporated herein by reference).

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryotic cells. Suitableprokaryotes for this purpose include eubacteria, such as Gram-negativeor Gram-positive organisms, for example, Enterobacteriaceae such asEscherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus,Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratiamarcescans, and Shigella, as well as Bacilli such as B. subtilis and B.licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710published 12 Apr. 1989), Pseudomonas such as P. aeruginosa, andStreptomyces. One preferred E. coli cloning host is E. coli 294 (ATCC31,446), although other strains such as E. coli B, E. coli X1776 (ATCC31,537), and E. coli W3110 (ATCC 27,325) are suitable. These examplesare illustrative rather than limiting.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for polypeptideencoding vectors. Saccharomyces cerevisiae, or common baker's yeast, isthe most commonly used among lower eukaryotic host microorganisms.However, a number of other genera, species, and strains are commonlyavailable and useful herein, such as Schizosaccharomyces pombe;Kluyveromyces hosts such as, e.g., K lactis, K. fragilis (ATCC 12,424),K bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K.marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida;Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces suchas Schwanniomyces occidentalis; and filamentous fungi such as, e.g.,Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A.nidulans and A. niger.

Suitable host cells for the expression of glycosylated polypeptide arederived from multicellular organisms. Examples of invertebrate cellsinclude plant and insect cells. Numerous baculoviral strains andvariants and corresponding permissive insect host cells from hosts suchas Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedesalbopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyxmori have been identified. A variety of viral strains for transfectionare publicly available, e.g., the L-1 variant of Autographa californicaNPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be usedas the virus herein according to the present invention, particularly fortransfection of Spodoptera frugiperda cells. Plant cell cultures ofcotton, corn, potato, soybean, petunia, tomato, and tobacco can also beutilized as hosts.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) has become a routineprocedure. Examples of useful mammalian host cell lines include, but arenot limited to, monkey kidney CV1 cells transformed by SV40 (COS-7, ATCCCRL 1651); human embryonic kidney cells (293 or 293 cells subcloned forgrowth in suspension culture, Graham et al., J. Gen Virol. 36:59(1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamsterovary cells/-DHFR(CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci.383:44-68 (1982)); MRC 5 cells; FS4 cells; and human hepatoma cells (HepG2).

Host cells are transformed with the above-described expression orcloning vectors for polypeptide production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

The host cells used to produce the polypeptide of this invention may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal.Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762;4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. No. Re.30,985 may be used as culture media for the host cells. Any of thesemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleotides (such as adenosine and thymidine),antibiotics (such as GENTAMYCIN™ drug), trace elements (defined asinorganic compounds usually present at final concentrations in themicromolar range), and glucose or an equivalent energy source. Any othernecessary supplements may also be included at appropriate concentrationsthat would be known to those skilled in the art. The culture conditions,such as temperature, pH, and the like, are those previously used withthe host cell selected for expression, and will be apparent to theordinarily skilled artisan.

When using recombinant techniques, the polypeptide can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the polypeptide is produced intracellularly, as a first step,the particulate debris, either host cells or lysed cells (e.g. resultingfrom homogenization), is removed, for example, by centrifugation orultrafiltration. Where the polypeptide is secreted into the medium,supernatants from such expression systems are generally firstconcentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit.

The polypeptide is then subjected to one or more purification steps,including the ion exchange chromatography method as described herein.Examples of additional purification procedures which may be performedprior to, during, or following the ion exchange chromatography methodinclude fractionation on a hydrophobic interaction chromatography (e.g.on phenyl sepharose), ethanol precipitation, isoelectric focusing,Reverse Phase HPLC, chromatography on silica, chromatography on HEPARINSEPHAROSE™, further anion exchange chromatography and/or further cationexchange chromatography, chromatofocusing, SDS-PAGE, ammonium sulfateprecipitation, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography (e.g. using protein A, protein G,an antibody, a specific substrate, ligand or antigen as the capturereagent).

Ion exchange chromatography is performed as described herein. A decisionis first made as to whether an anion or cation exchange resin is to beemployed. In general, a cation exchange resin may be used forpolypeptides with pI's greater than about 7 and an anion exchange resinmay be used for polypeptides with pI's less than about 7.

The anion or cation exchange resin is prepared according to knownmethods, following the manufacturer's instructions. Usually, anequilibration buffer is passed through the ion exchange resin prior toloading the composition comprising the polypeptide of interest and oneor more contaminants onto the resin. Conveniently, the equilibrationbuffer is the same as the loading buffer, but this is not required. Thecomposition of the various buffers used for the chromatography maydepend in part on whether a cation or anion exchange resin is employed.

Following equilibration, an aqueous solution comprising the polypeptideof interest and contaminant(s) is loaded onto the cation exchange resinusing a buffer that is at a pH and/or conductivity such that thepolypeptide and the contaminant bind to the cation exchange resin. Asdiscussed above, the equilibration buffer may be used for loading. In apreferred embodiment, the equilibration buffer is at a first lowconductivity (e.g. from about 4 to about 5 mmhos) during loading. Anexemplary pH for the equilibration buffer is about 5.5.

The amount of the polypeptide of interest loaded onto the resin maydepend on a variety of factors, including, for example, the capacity ofthe resin, the desired yield, and the desired purity. Preferably, fromabout 1 mg of protein/ml of resin to about 100 mg of protein/ml ofresin, more preferably from about 10 mg/ml to about 75 mg/ml and evenmore preferably from about 15 mg/ml to about 45 mg/ml of the polypeptide(e.g. of a full-length antibody) is loaded on the ion exchange resin.

After loading, the cation exchange resin is washed. During the washprocess, wash buffer is passed over the resin. The composition of thewash buffer is typically chosen to elute as many contaminants aspossible from the resin without eluting a substantial amount of thepolypeptide of interest. This may be achieved by using a wash bufferwith an increased conductivity or pH, or both, compared to theequilibration buffer. The composition of the was buffer may be constantor variable over the wash process, as described below.

In one embodiment, the wash buffer comprises equilibration buffer inwhich the salt concentration has been increased. The salt concentrationmay be increased by any method known in the art. In the preferredembodiment the wash buffer is a mixture of equilibration buffer andelution buffer. In this case, the desired salt concentration in the washbuffer is achieved by increasing the percentage of the higher saltbuffer in the wash buffer. The elution buffer typically has a highersalt concentration and conductivity than the equilibration buffer. Forexample, in the preferred embodiment the elution buffer preferably has aconductivity of between about 8 mS/cm and about 10 mS/cm, morepreferably between about 8.5 mS/cm and 9.5 mS/cm, while theequilibration buffer has a conductivity of between about 4 and 6 mS/cm,more preferably between about 4.5 and 5.5 mS/cm. Thus, as the percentageof elution buffer is increased, the salt concentration and theconductivity of the wash buffer increase.

The increase in salt concentration from the equilibration buffer to theinitial wash buffer may be step-wise or gradual as desired. The amountof the initial increase will depend upon the desired conductivity of thewash buffer at the start of the wash process.

Preferably, the initial increase in the salt concentration is achievedby a stepwise increase in the percentage of elution buffer in the washbuffer. One of skill in the art will be able to determine the amount ofthe increase in elution buffer based on the salt concentration of theequilibration buffer, the salt concentration of the elution buffer andthe desired conductivity. The increase is preferably from about 0%elution buffer to an initial percentage of between about 10% and 50%elution buffer, more preferably to between about 20% and 30% elutionbuffer and still more preferably to about 25% elution buffer. In thepreferred embodiment, the initial increase is to an initial percentageof about 26% elution buffer.

A wash buffer with a fixed salt concentration may be used for the entirewash. In this case the composition of the wash buffer will not varysignificantly from the initial composition for the duration of the wash.Preferably, however, a gradient wash is used, in which the compositionof the wash buffer changes over the course of the wash process.

In one embodiment a linear salt concentration gradient wash is used. Inthis wash process, the salt concentration of the wash buffer changes ata constant rate, generally increasing from a first initial concentrationto a higher second concentration, as the wash progresses. A linear saltconcentration gradient is preferably created by increasing thepercentage of elution buffer in the wash buffer at a constant rate. Therate of increase in the salt concentration of the wash buffer may bedescribed by the slope of the line formed by plotting the percentage ofelution buffer in the wash buffer against column volumes of wash bufferthat have passed over the column.

An exemplary linear gradient wash is depicted in FIG. 2. In this case, asingle slope defines the linear salt concentration gradient and thus therate of change in the salt concentration during the wash. The rate ofchange, and thus the slope, is chosen to achieve the greatest yield ofthe polypeptide of interest with the highest purity. One of skill in theart will be able to determine the optimum slope for a particularpolypeptide and load mass. In general, loads with a higher proteinconcentration will require a steeper slope, while smaller loads requirea shallower slope to achieve the desired yield and purity for a givenprotein.

In another embodiment, a multi-slope gradient wash is used. In this washprocess, a number of linear salt gradients with different slopes arecarried out consecutively. Thus, the salt concentration in the washbuffer passing over the column increases at a first rate for a firstportion of the wash, or segment, and at one or more additional rates forother defined portions, or segments of the wash. A three segment saltgradient is illustrated in FIG. 3 and described in more detail below.

Each linear salt gradient segment accounts for a fraction of the totalwash volume that passes over the resin. The proportion of the total washvolume accounted for by each particular gradient segment will varydepending on the number and duration of the segments.

One of skill in the art will recognize that the number of segments isnot limited in any way and will be chosen based on the particularcircumstances. Factors that will affect the preferred number of segmentsinclude, for example, the nature of the protein being eluted, the totalwash volume and the anticipated load range. For example, a multi-slopegradient wash allows a single wash protocol to be effective across awide column load range. Thus, if a variety of different load masses willbe purified using the same wash protocol, multiple segments will resultin more consistent yield and purity across the load range.

Each segment of a multi-segment wash preferably ends when apredetermined condition is met. For example when the concentration ofprotein in the flow through reaches a predetermined level, or when thewash buffer has reached a desired conductivity. In the preferredembodiment, each segment ends when the wash buffer comprises apredetermined percentage of elution buffer, and thus has a desiredconductivity.

In addition, if the wash comprises more than one segment, the slope ispreferably greater in the first segment than in any additional segments.As a result, the increase in the conductivity of the wash buffer will begreater in the first segment than in subsequent segments. If the saltconcentration is varied by increasing the percentage of elution bufferin the wash buffer, the increase in the percentage of elution buffer percolumn volume will be greater in the first segment than in subsequentsegments.

The gradient wash preferably ends when a predetermined amount of proteinis detected in the flow through. In the preferred embodiment, thegradient wash ends when the protein concentration in the flow throughreaches a level corresponding to an optical density measurement of 0.6at 280 nm.

The wash process is optionally completed by passing a fixed amount ofwash buffer over the column. This is referred to as the “end-wash delayvolume.” Following the last linear gradient segment, a fixed volume ofthe wash buffer with the highest conductivity from the gradient wash ispassed over the column. Using a larger end-wash delay volume increasesthe purity of the eluted protein but may lead to a somewhat decreasedyield. Conversely, decreasing the fixed volume leads to an increasedyield, but may produce a slight decrease in the purity of the recoveredprotein. Thus, the fixed volume may be chosen by the skilled artisan toachieve the desired yield and purity. Preferably the end-wash delayvolume is from 0 to 2 column volumes of the final wash buffer, morepreferably from 0.2 to 1 column volume.

In a preferred embodiment, a multi-slope gradient wash with threesegments is used. This embodiment is illustrated in FIG. 3. The firstsegment preferably accounts for about a third of the total wash volume,during which the percentage of elution buffer in the wash bufferincreases from an initial percentage of about 26% to about 50%, morepreferably about 54%, resulting in a corresponding increase in the saltconcentration and conductivity. About 5 column volumes of wash bufferare passed over the column during this first linear gradient segment.This represents a change of between about 5% and 6% elution buffer percolumn volume, as can be seen in the slope of the first segment in FIG.3.

A second linear gradient segment is begun by modifying the rate ofchange of the percentage of elution buffer in the wash buffer. In thepreferred embodiment the rate of change of the percentage of elutionbuffer is reduced to about 3.5% per column volume. The second segmentpreferably continues for approximately one sixth of the total washvolume. Thus, about 2 column volumes are passed over the column duringthe second segment. Preferably the second segment ends when thepercentage of elution buffer has increased to about 60%, more preferablyabout 61%.

In the third segment, the rate of increase of elution buffer is furtherreduced, preferably to about 2% per column volume, more preferably toabout 2.13% per column volume. The third segment is the longest of thethree and accounts for about half of the total wash volume, with about 6column volumes of wash buffer passing over the column. The third segmentends when an OD of 0.6 is measured in the flow through. Over the entiregradient wash, the percentage of elution buffer will have increased toabout 75%, more preferably about 74% when an OD of 0.6 is achieved.

Following the wash process, a predetermined amount of equilibrationbuffer is optionally passed over the column. Preferably from 0 to 2column volumes of equilibration buffer are passed over the column. Morepreferably 1 column volume of equilibration buffer is passed over thecolumn.

The desired polypeptide molecule is subsequently eluted from the ionexchange resin. This is achieved using an elution buffer that has a pHand/or conductivity such that the desired polypeptide no longer binds tothe ion exchange resin and therefore is eluted therefrom. In thepreferred embodiment, the conductivity of the elution buffer exceedsthat of the equilibration buffer. Alternatively, or in addition, the pHof the elution buffer may be increased relative to the equilibrationbuffer (for example, the pH of the elution buffer may about 6.0). Thechange in conductivity and/or pH from the wash buffer to the elutionbuffer may be step-wise or gradual, as desired. As discussed above, theelution buffer preferably has a conductivity of between about 8 mS/cmand about 10 mS/cm, more preferably between about 8.5 mS/cm and 9.5mS/cm. Hence, the desired polypeptide is retrieved from the cationexchange resin at this stage in the method.

The changes in conductivity are generally as described above withrespect to both a cation exchange resin and an anion exchange resin. Oneof skill in the art will be able to optimize the methods for either typeof resin.

In the preferred embodiment of the invention, a single parameter (i.e.either conductivity or pH) is changed to achieve elution of both thepolypeptide and contaminant, while the other parameter (i.e. pH orconductivity, respectively) remains about constant. For example, whilethe conductivity of the various buffers may differ, the pH's thereof maybe essentially the same.

In an optional embodiment of the invention, the ion exchange resin isregenerated with a regeneration buffer after elution of the polypeptide,such that the column can be re-used. Generally, the conductivity and/orpH of the regeneration buffer is/are such that substantially allcontaminants and the polypeptide of interest are eluted from the ionexchange resin. Generally, the regeneration buffer has a very highconductivity for eluting contaminants and polypeptide from the ionexchange resin.

The method herein is particularly useful for resolving a polypeptidemolecule of interest from at least one contaminant, where thecontaminant and polypeptide molecule of interest differ only slightly inionic charge. The method may also be used, for example, to resolve apolypeptide from a glycosylation variant thereof, e.g. for resolving avariant of a polypeptide having a different distribution of sialic acidcompared to the non-variant polypeptide.

The polypeptide preparation obtained according to the ion exchangechromatography method herein may be subjected to additional purificationsteps, if necessary. Exemplary further purification steps have beendiscussed above.

Optionally, the polypeptide is conjugated to one or more heterologousmolecules as desired. The heterologous molecule may, for example, be onewhich increases the serum half-life of the polypeptide (e.g.polyethylene glycol, PEG), or it may be a label (e.g. an enzyme,fluorescent label and/or radionuclide) or a cytotoxic molecule (e.g. atoxin, chemotherapeutic drug, or radioactive isotope etc).

A therapeutic formulation comprising the polypeptide, optionallyconjugated with a heterologous molecule, may be prepared by mixing thepolypeptide having the desired degree of purity with optionalpharmaceutically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),in the form of lyophilized formulations or aqueous solutions.“Pharmaceutically acceptable” carriers, excipients, or stabilizers arenontoxic to recipients at the dosages and concentrations employed, andinclude buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptide; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). ThehumMAb4D5-8 antibody of particular interest herein may be prepared as alyophilized formulation, e.g. as described in WO 97/04801; expresslyincorporated herein by reference.

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Such molecules are suitably present in combination in amounts that areeffective for the purpose intended. For example, for an anti-HER2antibody a chemotherapeutic agent, such as a taxoid or tamoxifen, may beadded to the formulation.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulation to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the polypeptide variant, which matricesare in the form of shaped articles, e.g., films, or microcapsule.Examples of sustained-release matrices include polyesters, hydrogels(for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acidand γ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT™ (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.

The polypeptide purified as disclosed herein or the compositioncomprising the polypeptide and a pharmaceutically acceptable carrier isthen used for various diagnostic, therapeutic or other uses known forsuch polypeptides and compositions. For example, the polypeptide may beused to treat a disorder in a mammal by administering a therapeuticallyeffective amount of the polypeptide to the mammal.

The following examples are offered by way of illustration and not by wayof limitation. The disclosures of all citations in the specification areexpressly incorporated herein by reference.

EXAMPLES

Full length human IgG rhuMAb HER2 (humAb4D5-8 in Carter et al. Proc.Natl. Acad. Sci. 89: 4285-4289 (1992) comprising the light chain aminoacid sequence of SEQ ID NO:1 and heavy chain amino acid sequence of SEQID NO:2) was produced recombinantly in CHO cells. Following proteinproduction and secretion to the cell culture medium, the CHO cells wereseparated from the cell culture medium by tangential flow filtration(PROSTACK™). Protein A chromatography was then performed by applying theHarvested Cell Culture Fluid (HCCF) from the CHO cells directly to anequilibrated PROSEP A™ column (Bioprocessing, Ltd).

Following Protein A chromatography, cation exchange chromatography wasperformed using a sulphopropyl (SP-SEPHAROSE FAST FLOW XL™ (SPXLFF)column (Pharmacia) to further separate the desired anti-HER2 antibodymolecule. An SPXLFF™ column was packed. The dimensions were: 100 cmdiameter and 35 cm bed height. The conductivity of the pool was reducedby the addition of an equal volume of sterile water for injection(SWFI).

The chromatography runs for these studies were performed withPharmacia's UNICORN™ FPLC system. A number of chromatography runs wereperformed with load densities of 15, 30, and 45 mg of rhuMAb HER2 per mLof SPXLFF resin. In addition, the delay volume following the gradientwash was varied. Delay volumes of 1.0, 0.8, 0.6 and 0.4 column volumesfollowing the gradient were tested.

The protein concentration of each chromatography fraction was determinedby spectrophometric scans of each sample. The results were used tocalculate product recovery yields. The extinction coefficient for rhuMAbHER2 is 1.45. Calculations used to derive the results are:

${{Protein}\mspace{14mu}{{Concentration}\left( {{mg}\text{/}{mL}} \right)}} = {\frac{280\mspace{14mu}{nm}}{1.45} \times {Dilution}\mspace{14mu}{Factor}}$Protein  Mass(mg)  in  Each  Fraction   = Protein  Concentration(mg/mL) × Fraction  Volume  (mL)${{Yield}(\%)} = {\frac{{Fraction}\mspace{14mu}{{Mass}({mg})}}{{Total}{\mspace{11mu}\;}{{Mass}({mg})}} \times 100}$

Deamidated and other acidic variants of rhuMAb HER2 were produced whenthe antibody was made by recombinant DNA technology. Fractions from eachof the study chromatographies were tested for the relative amount ofvariant antibody by Dionex HPIEC chromatography. The deamidated andother acidic variants constituted from about 15 to about 20% of thecomposition obtained from the initial Protein A chromatography. It wasdiscovered that the ion exchange methods described below could be usedto substantially reduce the amount of deamidated and other acidicvariants in the anti-HER2 composition, typically by about 50% or more.

Example 1

The SPXLFF column was prepared for load by sequential washes withregeneration buffer (0.5 N NaOH) followed by equilibration buffer (30 mMMES/45 mM NaCl, pH 5.6). The column was then loaded with Protein A pooladjusted to a pH of 5.60±0.05 and a conductivity of 5.8±0.2 mmhos. Thecolumn was washed using a linear salt gradient, essentially as depictedin FIG. 2. Prior to beginning the linear gradient, elution buffer (30 mMMES, 100 mM NaCl, pH 5.6) was mixed with equilibration buffer to producean initial wash buffer that comprised about 26% elution buffer. Theslope of the linear gradient was varied in different experiments, withrates of increase of salt concentration of 1 mM NaCl/Column volume (CV),2 mM/CV and 3 mM/CV used. The linear gradient continued until an OD of0.6 at 280 nm was measured in the flow through. The column was thenwashed with 1 CV of the ultimate wash buffer.

rhuMAb HER2 was then eluted from the column with elution buffer (30 mMMES/100 mM NaCl, pH 5.6). Following elution, the column was regeneratedwith regeneration buffer (0.5 N NaOH).

As can be seen in Table 1, the step yield varied as a result of slopeand load mass.

TABLE 1 Step Yield as a Function of Gradient Slope and Load Mass Slope15 mg/ml 30 mg/ml 45 mg/ml 1 mM/CV 83% 85% 90% 2 mM/CV 81% 81% 84% 3mM/CV 69% 73% 77%

The purity was measured in terms of acidic variants by Dionex HPIEC. Ascan be seen in Table 2, the purity generally increased with increasinggradient slope. The load comprised 17% acid variants in all cases.

TABLE 2 Dionex HPIEC Purity Results; % Acidic Variants Slope 15 mg/ml 30mg/ml 45 mg/ml 1 mM/CV 7% 12% 14% 2 mM/CV 4% 7% 10% 3 mM/CV 2% 4% 7%

Example 2

An SPXLFF column, as described above, was prepared for load bysequential washes with regeneration buffer (0.5 N NaOH) followed byequilibration buffer (30 mM MES/70 mM Na/HOAc, pH 5.5). The column wasthen loaded with Protein A pool adjusted to a pH of 5.60±0.05 and aconductivity of 5.8±0.2 mmhos.

Following loading, the column was washed using a multi-slope saltgradient with three distinct segments, each having a progressivelyshallower slope, essentially as illustrated in FIG. 3. The gradientparameters are shown in Table 3 below.

The wash began with an initial stepwise increase in the saltconcentration of the equilibration buffer to form the initial washbuffer. The initial wash buffer was created by mixing elution buffer (30mM MES, 145 mM Na/HOAc, pH 5.5) with equilibration buffer to produce abuffer that comprised 26% elution buffer. During the first lineargradient segment the column was washed with approximately 4.9 columnvolumes of wash buffer, during which the percentage of elution buffer inthe wash buffer increased at a rate of about 5.71% per column volume.Thus, at the end of the first linear gradient segment, the wash buffercomprised 54% elution buffer.

During the second linear gradient segment 2.0 column volumes of washbuffer were passed over the column. During this segment the percentageof elution buffer increased at a rate of 3.5%. Thus, at the end of thesecond segment the wash buffer comprised 61% elution buffer.

Finally, during the third linear gradient segment 6.1 column volumes ofwash buffer were passed over the column, with the percentage of elutionbuffer in the wash buffer increasing at a rate of 2.13% per columnvolume. The third linear gradient segment ended when an OD of 0.6 at 280nm was measured in the flow through. At this point the wash buffercomprised 74% elution buffer.

Following the third linear gradient segment a fixed volume of the finalwash buffer (i.e. 74% elution buffer) was passed over the column. Fixedvolumes of 0.8, 0.6 and 0.4 column volumes were used in differentexperiments.

rhuMAb HER2 was then eluted from the column with elution buffer (30 mMMES/145 mM Na/HOAc, pH 5.5). Following elution, the column wasregenerated with regeneration buffer (0.5 N NaOH).

TABLE 3 Gradient Parameters % elution buffer Total Wash Volume Slope(end of at End of Segment Total Segment % elution segment) (ColumnVolumes) Volume (CV) buffer/CV Gradient 26% 0 0 Start Segment 1 54% 4.94.9 5.71% Segment 2 61% 6.9 2.0 3.50% Segment 3 74% 13 6.1 2.13%

The effect of rhuMAb HER2 load and end-wash delay volume on productrecovery, and product quality was evaluated. The results presented inTables 4 and 5 show that it is possible to achieve consistent yield andacidic variant removal over a wide load range by using a multi-slopegradient wash. In addition, the results demonstrate that the trade offbetween yield and purity can be fine tuned as desired by adjusting theend-wash delay volume after an OD of 0.6 is achieved.

As can be seen in Table 4, for a given end-wash delay volume, the stepyield does not vary significantly. A larger end-wash delay volumedecreases the yield somewhat at all load masses. However, as shown inTable 5, increasing the end-wash delay volume increases the purity ofthe eluted protein. Thus, one of skill in the art will be able to selectan end-wash delay volume that achieves the desired yield and purity.

TABLE 4 Step Yield as a Function of Wash Delay Volume and Load Mass 15mg/ml 30 mg/ml 45 mg/ml 0.8 CV 79% 80% 81% 0.6 CV 80% 81% 83% 0.4 CV 83%84% 85%

TABLE 5 Dionex HPIEC Purity; % Acidic Variants 15 mg/ml 30 mg/ml 45mg/ml 0.8 CV 6% 7% 9% 0.6 CV 8% 10% 11% 0.4 CV 10% 11% 12% Based on loadof 19% acidic variants.

By using a multi-slope gradient with three segments, each having aprogressively shallower slope, consistent yield and purity can beachieved across a wide load range. Between the ranges of 15 to 45 mg ofantibody per mL of resin, there is little variation in the yield or thequality of rhuMAb HER2 recovered in the elution pool for a given columnwash delay volume.

What is claimed is:
 1. A method for purifying an antibody from acomposition comprising the antibody and a contaminant, which methodcomprises the following steps performed sequentially: (a) binding theantibody to a cation exchange material with an equilibration buffer at afirst conductivity; (b) washing the cation exchange material with a washbuffer, wherein the conductivity of the wash buffer increases from asecond conductivity that is higher than the first conductivity to athird conductivity during the washing; (c) passing a fixed volume ofwash buffer at the third conductivity over the cation exchange material;and (d) eluting the antibody from the cation exchange material with anelution buffer at a fourth conductivity that is higher than the thirdconductivity.
 2. The method of claim 1 wherein the cation exchange resincomprises sulphopropyl immobilized on agarose.
 3. The method of claim 1wherein the conductivity of the wash buffer increases at a constant ratefrom the second conductivity to the third conductivity.
 4. The method ofclaim 1 wherein the conductivity of the wash buffer increases at two ormore different rates from the second conductivity to the thirdconductivity.
 5. The method of claim 4 wherein the conductivity of thewash buffer increases at a first rate for a first segment of thewashing, at a second rate for a second segment of the washing and at athird rate for a third segment of the washing.
 6. The method of claim 5wherein the wash buffer comprises a mixture of equilibration buffer andelution buffer.
 7. The method of claim 6 wherein the conductivity of thewash buffer is increased by increasing the proportion of elution bufferin the wash buffer.
 8. The method of claim 7 wherein the proportion ofelution buffer in the wash buffer increases at a constant rate of about6% during the first segment, at a constant rate of about 3.5% during thesecond segment and at a constant rate of about 2% during the thirdsegment.
 9. The method of claim 7 wherein the proportion of elutionbuffer in the wash buffer increases from about 26% to about 54% duringthe first segment, from about 54% to about 61% during the second segmentand from about 61% to about 74% during the second segment.
 10. Themethod of claim 5 wherein the cation exchange material is washed withabout 5 column volumes of wash buffer in the first segment, about 2column volumes of wash buffer in the second segment and about 6 columnvolumes of wash buffer in the third segment.
 11. The method of claim 1wherein the conductivity of the wash buffer is increased by increasingthe proportion of elution buffer in the wash buffer.
 12. The method ofclaim 1 wherein the conductivity of the wash buffer is increased byincreasing the salt concentration therein.
 13. The method of claim 1wherein the fixed volume of wash buffer passed over the cation exchangematerial in step (c) is between about 0.4 column volumes and about 1.0column volumes.
 14. The method of claim 1 further comprising washing theion exchange material with a regeneration buffer after step (d).